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BMC Developmental Biology

Open Access

Dynamic expression of Dab2 in the mouse embryonic central nervous system

  • Kwok-Kuen Cheung1, 2,
  • Samuel C Mok3,
  • Payam Rezaie4, 5 and
  • Wood Yee Chan1Email author
BMC Developmental Biology20088:76

https://doi.org/10.1186/1471-213X-8-76

Received: 27 March 2008

Accepted: 04 August 2008

Published: 04 August 2008

Abstract

Background

Dab2, one of two mammalian orthologs of Drosophila Disabled, has been shown to be involved in cell positioning and formation of visceral endoderm during mouse embryogenesis, but its role in neuronal development is not yet fully understood. In this report, we have examined the localization of the Dab2 protein in the mouse embryonic central nervous system (CNS) at different developmental stages.

Results

Dab2 protein was transiently expressed in rhombomeres 5 and 6 of the developing hindbrain between E8.5 and E11.5, and in the floor plate of the neural tube from E9.5 to E12.5, following which it was no longer detectable within these regions. Dab2 protein was also identified within circumventricular organs including the choroid plexus, subcommissural organ and pineal gland during their early development. While Dab2 was still strongly expressed in the adult choroid plexus, immunoreactivity within the subcommissural organ and pineal gland was lost after birth. In addition, Dab2 was transiently expressed within a subpopulation of Iba1-positive mononuclear phagocytes (including presumed microglial progenitors) within the neural tube from E10.0 and was lost by E14.5. Dab2 was separately localized to Iba1 positive cells from E9.5 and subsequently to F4/80 positive cells (mature macrophage/myeloid-derived dendritic cells) positioned outside the neural tube from E12.5 onwards, implicating Dab2 expression in early cells of the mononuclear phagocyte lineage. Dab2 did not co-localize with the pan-neuronal marker PGP9.5 at any developmental stage, suggesting that Dab2 positive cells in the developing CNS are unlikely to be differentiating neurons.

Conclusion

This is the first study to demonstrate the dynamic spatiotemporal expression of Dab2 protein within the CNS during development.

Background

Dab2 was first identified as DOC-2 which is differentially expressed in ovarian carcinoma and cell lines [1]. It was later characterized as a tumor suppressor in various types of tumors [27]. Subsequent studies indicated Dab2 as one of two mammalian orthologs of the Drosophila Disabled (dDab) [811], a family member of the Disabled proteins involved in neurogenesis, oncogenesis, cellular signaling and embryonic development. Three different spliced forms of Dab2 cDNAs encoding variants p96, p93 and p67 were previously identified from a mouse macrophage library [12]. Of these, p96 was isolated as a mitogenic responsive phosphoprotein with possible function in the macrophage CSF-1 signal transduction pathway [12, 13]. The complete genomic DNA sequence of the mouse Dab2 gene and its chromosomal localization at 15A2 were reported [14].

Sequence analyses [1, 11, 14] indicated Dab2 as an adaptor protein involved in signal transduction. The N-terminus of the protein contains a highly conserved phosphotyrosine interacting domain (PID or PTB) which interacts with proteins phosphorylated on tyrosine residue. Within its C-terminus, the proline-rich domain exhibits multiple SH3 binding motifs capable of interacting with SH3-containing signaling molecules such as Grb2. Signaling pathways involving Dab2 have not been fully characterized. However, there is evidence to suggest that Dab2 is an essential component of the transforming growth factor β (TGFβ) signaling pathway (involved in transmission of TGFβ signaling from TGFβ receptors to the Smad family of transcriptional activators) [15]. Dab2 has also been shown to act as a regulator of the Ras/mitogen-activated protein kinases (MAPK) pathway [16, 17], Src activity [7] and canonical Wnt/β-catenin-induced signaling [18].

Considering that molecular interactions through such signaling pathways are important for normal growth and differentiation of embryonic structures, it has been proposed that Dab2 may also be involved in a number of different developmental processes [19]. For example, expression of Dab2 is upregulated upon retinoic acid treatment [20] or during the normal differentiation of F9 cells, a multipotent embryonic carcinoma cell line [21], into primitive endoderm-like cells [22]. Cho et al. also showed that the temporal expression pattern of the murine Dab2 gene coincided with the initiation pattern of retinoic acid synthesis in embryonic mice [20]. Furthermore, gene targeting studies have demonstrated that Dab2 is a direct and specific downstream target of the zinc finger transcription factor GATA-6 in the visceral endoderm [23]. In the absence of Dab2, the visceral endoderm becomes disorganized, resulting in growth failure of the inner cell mass. The Dab2-deficient embryo then fails to gastrulate and ceases development at around embryonic day 6.0 to 6.5 [19, 23, 24]. In addition to the visceral endoderm, Dab2 mRNA is also expressed in the branchial arch mesenchyme and septum transversum during development [23]. A recent study showed that Dab2 is required for TGFβ-induced epithelial-to-mesenchymal transition [25], which is an important multi-step process necessary for the proper development of many early embryonic structures including the neural crest, the primitive streak and somites [2628].

Another mammalian ortholog of the Drosophila Disabled is Dab1, a related gene which also contains PID in its N-terminus. It has already been demonstrated that the Dab1 protein is important in regulating the positioning of migrating neurons during mammalian neurogenesis [2931]. Mouse Dab1 is expressed in cortical neurons, and targeted disruption of Dab1 resulted in abnormal neuronal layering in the developing cerebral cortex and cerebellum, together with an indistinct dentate gyrus and CA1 and CA3 regions of the hippocampus [29]. Such defects partly resulted from a failure of Dab1-/- neurons to detach from radial glia during the late stage of neuronal migration [30]. Despite the functional similarity of Dab1 and Dab2 proteins in cell positioning during development, it is not known whether Dab2 also play key roles during the development of the embryonic central nervous system (CNS), although it has recently been shown that Dab2 inhibits NGF-mediated neurite outgrowth in vitro [32]. Whether the expression of Dab2 is regulated in a developmental fashion has not been determined previously. Therefore in this investigation our primary aim was to characterize the spatiotemporal patterns of expression and the cellular localization of Dab2 protein within the embryonic murine CNS as an initial step towards further understanding the developmental roles of Dab2.

Results

Dab2 immunoreactivity in the floor plate and rhombomeres

Dab2 immunoreactivity was first observed in the neuroepithelium of the prospective hindbrain region at E8.5 (8–10 somites stage) (Fig 1A, A'). The expression was observed as a small positive band near the caudal margin of the closing anterior neuropore, corresponding to the position of rhombomeres 5 and 6 (Fig 1A, A'). This was regionally restricted, as other regions of the neuroepithelium lacked Dab2 expression. At E9.0 and E9.5, the neuroepithelium of rhombomere 6 was strongly Dab2 positive in its entire extent (Fig 1B, C, C', E, F), whereas only the ventral part of rhombomere 5 showed immunoreactivity (Fig 1F, G, G"). In addition to rhombomeres 5 and 6, Dab2 was also expressed in a narrow strip of the ventral neural tube extending from the mesencephalic flexure down to the spinal neural tube (Fig 1B, F, G). Sections through the spinal neural tube revealed Dab2 expression restricted to the floor plate (Fig 1I, I'). Dab2 expression in the floor plate was initially detected in the rostral part of the spinal cord and gradually shifted in a rostral-to-caudal manner to the caudal spinal cord (Fig 1N, compare Fig 1I with Fig 1I'). Weak Dab2 immunoreactivity was also detected in the caudal part of the notochord (Fig 1I'). By E10.5, the Dab2 protein expression was significantly down-regulated in these rhombomeres (Fig 1H) and became undetectable by E11.5. Likewise, Dab2 expression was progressively down-regulated in the floor plate of the neural tube from E10.5 onwards. Down-regulated expression of Dab2 in the floor plate, also occurred in a rostral-to-caudal direction along the spinal cord (Fig 1N, also compare Fig 1I–K with Fig 1I'–K'). Dab2 immunoreactivity in the floor plate became undetectable by E12.5 (Fig 1M). Negative controls did not show any immunoreactive signals (Fig 1D, G').
Figure 1

Dab2 immunoreactivity in the neural tube. In whole mount preparations (A-D), tissues surrounding the neural tube, including the otic vesicle (asterisks in C) had been removed by microdissection. At E8.5, the neural tube (A: dorsal view; A': lateral view) already shows Dab2 expression in rhombomeres 5 and 6 (arrowheads). At E9.0 (B: lateral view) and E9.5 (C: dorsal view; C': lateral view), expression within rhombomeres 5 and 6 (r5 and r6, arrows in C') remains strong, and spreads along the floor plate of the neural tube (arrowheads in B). The lack of immunoreactivity in the pre-adsorption control (D) confirms the specificity of the Dab2 immunoreaction. An oblique section (E) and longitudinal sections (F, G, G") through E9.5 embryos demonstrate strong Dab2 expression in the whole span of r6 and weak immunoreactivity in the ventral part of r5 (G" shows the high magnification of the immunoreactive r5 and r6 in G). The pre-adsorption control section (G') adjacent to the section shown in G does not exhibit any immunoreactivity. The ventral expression of Dab2 in the floor plate of the neural tube extends rostrocaudally from the mesencephalic flexure (arrow, F) to the caudal spinal cord (arrow, G). At E10.5, Dab2 immunoreactivity becomes weak in r6 (arrow, H) (Hb; hindbrain; 4V: fourth ventricle) and absent in r5. Dab2 immunoreactivity along the floor plate of the spinal cord (arrowheads, I to M) is also observed in transverse sections through the rostral (I, J, K, L, M) and caudal spinal cord (I', J', K') at different developmental stage. Also note the weak Dab2 staining in the notochord (arrow, I'). Insets in I to M represent the high magnification of the floor plate shown in corresponding figures. The schematic diagram (N) summarizes changes in Dab2 expression along the floor plate of the neural tube from E9.5 to E12.5. The dotted line indicates the axial level of the mesencephalic flexure and the shading represents Dab2 immunoreactivity observed in the floor plate of the neural tube. Arrowheads in N indicate regions of the neural tube from which sections shown in I, I', K, K', L, M were taken. Note that the onset and down-regulation of Dab2 expression follow a rostral-to-caudal sequence. Scale bar: 100 μm for I; 50 μm for G' and I', 25 μm for insets of I-L, and 200 μm for other panels.

Dab2 immunoreactivity in the developing choroid plexus

Dab2 protein was detected in the roof plate of the hindbrain between E9.5 and E11.5 (Fig 2A, B, B', C, C'). At E12.5, the developing choroid plexus of the lateral ventricles and the fourth ventricle (Fig 2D, D') were Dab2 immunoreactive. The choroid plexus of the third ventricle, which developed slightly later, was also positive for Dab2 (data not shown). However, the neuroepithelium adjacent to the choroid plexus was clearly negative and a distinct boundary between the immunoreactive choroid plexus and the adjacent neuroepithelium which lacked staining, was easily discernible (Fig 2B–D). The entire choroid plexus, including most of the epithelial cells that lined the ventricles and their underlying mesenchyme, was Dab2 positive (Fig 2D-F, 2D'). More intense expression was associated with the apical aspect (or luminal surface, and associated microvilli) as compared to the basal aspect of epithelial cells (Fig 2D'). The choroid plexus continued to express the Dab2 protein in adulthood, and consequently, became the most prominent Dab2 positive structure in the adult CNS (Fig 2G), with the exception that the intensity and localization of Dab2 expression was reversed: in contrast to the embryonic choroid plexus, which showed strong Dab2 immunoreactivity on the luminal (apical) side of the epithelium (Fig 2D'), the luminal surface of the epithelial cells in the adult choroid plexus showed weaker immunoreactivity compared to the basal surface (Fig 2G).
Figure 2

Dab2 immunoreactivity in the developing choroid plexus, subcommissural organ and pineal gland. Sections through the hindbrain (Hb) at stages from E9.5 to E11.5 (A to C; B' and C' represent the high magnification of the boxes in B and C, respectively) show Dab2 expression in the roof plate of the hindbrain (arrows) and on scattered round cells (mononuclear phagocytes). Note that the adjacent hindbrain neuroepithelium is negative for Dab2 staining (arrowheads in B, C). Sections through the hindbrain (Hb) at stages from E12.5 to adulthood (D to G, D' is the higher magnification of the choroid plexus shown in D) show Dab2 staining in the choroid plexus (e.g. arrow in D) of the fourth ventricle (4V). Note the stronger Dab2 staining in the luminal (apical) surface of the epithelial cells as compared to the corresponding basal surface in embryos (arrowheads in D), but however, in the adult choroid plexus, the basal surface shows stronger Dab2 staining than the luminal (apical) surface of the epithelial cells (arrowheads in G). Also note the scattered and strongly Dab2 positive macrophages (arrows in D'). Sagittal sections show Dab2 staining in the subcommissural organ along the dorsal midline at different stages (H to K; H' and I' represent the higher magnification of the boxes in H and I, respectively). Note that Dab2 protein is expressed in the neuroepithelium of the subcommissural organ (sco) located in the roof plate of the diencephalon (Di). It lies underneath the posterior commissure (pc), anterior to the midbrain (Mb) (I', J, K). Also note the thin processes (arrows in I', J, K), which are also Dab2 positive, extending from the subcommissural organ into the posterior commissure. Coronal section (L) shows absence of Dab2 staining in the adult subcommissural organ. Double labeling of Dab2 (green, M) with PGP9.5 (red, M') shows that they are not co-localized (M"). Arrowheads in M indicate autofluorescence in blood cells. The sagittal section (N) shows Dab2 staining in the developing pineal gland (pg) along the dorsal midline at E16.5. Fb: forebrain; Mb: midbrain; Hb: hindbrain. Di: diencephalon. 4V: 4th ventricle; pc: posterior commissure; sco: subcommissural organ; pg: pineal gland. Scale bar: 50 μm.

Dab2 immunoreactivity within the developing subcommissural organ and pineal gland

The presumptive subcommissural organ (SCO) develops from the region of the roof plate of the diencephalon just anterior to the mesencephalon (Fig 2H, H'). As development progresses, the SCO neuroepithelium, which lies underneath (ventral to) the posterior commissure, became morphologically more distinct from the adjacent neuroepithelium (Fig 2I–K). Dab2 immunoreactive cells were detected in the developing SCO at E11.5 and the expression became progressively more prominent (Fig 2H–K, 2H'–I'), particularly in the cell layers immediately below the posterior commissure and also in the thin processes extending from the SCO to the posterior commissure (Fig 2I', 2J–K). The ventricular aspect of the SCO however showed only weak Dab2 immunoreactivity (Fig 2I', 2J–K). Double immunofluorescent labeling of Dab2 and PGP9.5, a pan-neuronal marker, was performed in order to determine whether Dab2 was expressed in neurons or nerve fibers that were closely associated with the SCO. In embryos at late stages (E14.5–E16.5) the SCO neuroepithelium was Dab2 positive but negative for PGP9.5, whereas the posterior commissure above the SCO was PGP9.5 positive but negative for Dab2 (Fig 2M–M"), indicating that Dab2-immunoreactive cells were unlikely to represent differentiating neurons. Dab2 expression was persistently detected in the SCO during prenatal development but was not found in the adult (Fig 2L). The pineal gland additionally showed Dab2 immunoreactivity at E16.5 (Fig 2N) but Dab2 was not detectable in the adult pineal gland (data not shown).

Dab2 immunoreactivity in developing mononuclear phagocytes (fetal macrophages and microglial progenitors)

Within the developing neural tube

Scattered but strongly Dab2-immunoreactive cells were detected within the neuroepithelium at E10.0 – E10.5 (Fig 3A). Double labeling with the Iba1 antibody (Fig 3A'), a mononuclear phagocyte marker expressed on cells of the macrophage and microglial cell lineages [3335], showed that the Dab2 protein was co-expressed with Iba1 protein (Fig 3A"). Dab2 immunoreactive cells did not express PGP9.5, indicating that they were not early differentiating neurons (Fig 3B). Although all Dab2-positive cells showed Iba1 immunoreactivity, cells that were Iba1 positive but Dab2 negative (Iba1+/Dab2-) were also observed (Fig 3C). Iba1+/Dab2+ cells resembled the early fetal macrophages previously reported in the developing CNS [35], several of which were found in the proximity of blood vessels at E10.5 (Fig 3D) and E11.5 (Fig 3E). However, their close relationship with blood vessels was lost at E12.5, at which time Iba1+/Dab2+ cells were found dispersed throughout the neural tube (Fig 3F). Iba1+/Dab2+ fetal macrophages were also frequently detected inside the ventricles, in regions close to the ventricular wall (Fig 3G). From E14.5 onwards, however, scattered Iba1+/Dab2+ cells were no longer detected within the neural tube, leaving only Iba1+/Dab2- cells (presumed to represent parenchymal microglial progenitors) within the neural tube (Fig 3H, H', H"). The endothelial lining of the brain microvasculature also expressed Dab2 from E11.5 onwards (Fig 3E, F, I). In the adult, however, Dab2 immunoreactivity persisted only in perivascular macrophages (also widely referred to as 'perivascular cells') associated with cerebral blood vessels, but was absent from the endothelial lining of these vessels (Fig 3J). Perivascular cell expression of Dab2 was prominent throughout the entire adult CNS. These cells were not uniform in shape but were either oval with small processes, or flat and elongated in shape (Fig 3J), as has been previously described [36, 37]. Unlike embryonic CNS, where cells closely apposed to the microvasculature expressed both Iba1 and Dab2 protein, Dab2 positive perivascular cells in the adult brain did not appear to co-express Iba1 protein (Fig 3J–K). Instead, double immunofluorescent labeling revealed that some, but not all, of these Dab2 immunopositive perivascular cells co-expressed F4/80 antigen, indicating that they were of the macrophage lineage (mature macrophages) (Fig 3L, L', L").
Figure 3

Dab2 immunoreactivity in mononuclear phagocytes. A-A": Double labeling (immunofluorescence staining) on a sagittal section through an E10.5 embryos shows co-localization of Dab2 (green, arrows in A) and Iba1 (red, arrows in A') in mononuclear phagocytes (yellow, arrows in A") within the neural tube (Ne, white outline), and in fetal macrophages outside the neural tube. B: Double labeling shows that Dab2 immunoreactivity (green, indicated by arrows) does not overlap with PGP9.5 immunoreactivity (red) within the neural tube (white outline) in a sagittal section through an E11.5 embryo. C: Double labeling at E12.5 shows Iba1+/Dab2- microglial progenitors (arrows, red) within the neural tube (Ne) and Iba1+/Dab2+ fetal macrophages (arrowhead) in the adjacent mesenchyme (mes).D-F: Sagittal sections show that Dab2+ cells (arrows) are often closely apposed to blood vessels (arrowheads) within the neural tube at E10.5 (D) and E11.5 (E), but more scattered by E12.5 (F). G: Some Dab2+ cells (arrowheads) are also observed closely associated with the roof of the fourth ventricle (4V). H-H": Double labeling for Dab2 (green, H) and Iba1 (red, H') at E14.5 shows that Dab2 is only expressed in Iba1+ fetal macrophages (arrows) in the mesenchyme (mf: mesencephalic flexure, white outline) outside the neural tube (Ne) but absent in Iba1+ microglial progenitors within the neural tube. I: At E16.5, Dab2 expression is detected in the wall of blood microvessels (arrowheads), but Dab2+ microglial progenitors are no longer detectable at this stage of development. J, K: Sections of an adult brain show Dab2+ perivascular cells (arrowheads in J) and Iba1+ microglia (arrows in K). L-L": A merged image (L") shows co-localization of F4/80 protein (red, L') in some (arrows in L") but not all of the Dab2+ (green, L) cells lying on the wall of cerebral microvessels (mv, white outline). M: A sagittal section of an E9.5 embryo shows Dab2+ cells in the cranial mensenchyme (mes) outside the neural tube (Ne). N-N": A merged image at E9.5 shows complete co-localization (yellow, brightfield, arrows in N") of Dab2 (green, arrows in N) and Iba1 (red, arrows in N') immunoreactivities in fetal macrophages outside the neural tube (Ne, white outline). O-O": A section through an E14.5 choroid plexus shows complete co-localization (yellow, O") of Dab2 (green, O) and F4/80 (red, O') immunoreactivities. Arrowheads in O' and O" indicate autofluorescence from red blood cells. DAPI counterstained in B, C, H", L". Scale bar: 50 μm.

Outside the developing neural tube

Scattered cells strongly positive for Dab2 were also detected within mesenchymal tissues outside the neural tube from E9.5 (Fig 3M), about half a day earlier than Iba1+/Dab2+ cells inside the neural tube. These Dab2 positive cells were also Iba1 positive (Fig 3N, N', N"). The macrophage differentiation marker, F4/80 antigen, began to be expressed weakly in these fetal macrophages located outside the neural tube at E12.5 and became prominent from E14.5 onwards. Double immunofluorescence staining for Dab2 and F4/80 showed that all Dab2 positive cells were also F4/80 immunoreactive from E14.5 onwards (Fig 3O, O', O"). In contrast to the transient, developmentally-restricted expression of Dab2 protein associated with mononuclear cells (microglial progenitors) located inside the neural tube, Dab2 protein continued to be persistently expressed in macrophages throughout the body into adulthood (data not shown).

Discussion

We have characterized in detail the expression pattern for Dab2 in the mouse embryonic central nervous system throughout development. Dynamic expression of Dab2 protein was detected in the neuroepithelium, blood vessels, circumventricular organs and mononuclear phagocytes (fetal macrophages, microglial progenitors), strongly suggesting that Dab2 is an important molecule during mammalian CNS and myeloid cell development. Unlike Dab1, which is expressed in cortical neurons, Dab2 expression is restricted to non-neuronal cells or immature neuroepithelial cells.

At E8.5, while signaling genes such as Hoxa1, Hoxa2, Hoxb1, kr and Krox20 are interacting to pattern the early hindbrain [38], Dab2 is expressed in presumptive rhombomeres 5 and 6 (r5-6). There are 7 to 8 rhombomeres, morphologically repeated compartments, transiently segregated along the anteroposterior axis of the vertebrate embryonic hindbrain. These compartments play pivotal roles in the specification and segregation of neurons and neural crest cells along the anteroposterior axis of the hindbrain [39]. It is well-established that genes with rhombomere-restricted expression (e.g. Hox genes) function to establish the identities of compartments [40]. Interestingly, it has been shown that Dab2 expression is significantly up-regulated in Hoxa1 knockout embryonic stem cells [41]. In the early hindbrain at E8.0 Hoxa1 is expressed in r4-6 and gradually restricted to r4 at E8.5 [38]. Such down-regulation correlates spatiotemporally with the onset of Dab2 expression within r5-6 at E8.5. A further understanding of the interactions between Dab2 and Hoxa1 will greatly enhance our knowledge of signaling cascades associated with hindbrain specification.

During development, dorsoventral patterning along the neural tube is controlled by the expression of sonic hedgehog (shh) [42]. shh is expressed by cells along the ventral midline of the embryonic forebrain, in the floor plate and the notochord of the hindbrain and spinal cord [43]. Since the encoded protein is in a secreted form, Shh protein expression displays a more diffuse pattern [43]. Dab2 protein expression appears to overlap with the region expressing shh mRNA in the floor plate and notochord at E9.5 but not the regions showing Shh protein [43]. Moreover, Dab2 protein expression is turned off much earlier in the floor plate (absent in E12.5) than for Shh in the ventral spinal cord at E15.5 [43]. The potential involvement of Dab2 in anteroposterior and dorsoventral patterning requires further investigation.

Dab2 protein was also detected within circumventricular organs including the choroid plexus, the subcommissural organ and the pineal gland. Dab2 may represent one of the earliest markers for the choroid plexus primordium in the roof plate of the hindbrain. Its persistent expression in the choroid plexus throughout life, however, indicates that Dab2 may not only be involved in the early formation of the choroid plexus but may also have a role in the transport of macromolecules across the epithelial cells of the choroid plexus to the cerebral ventricles. Evidence to support this hypothesis comes from studies on megalin, a well-known receptor for Dab2 [44]. Megalin mediates the transport of apolipoprotein J, Alzheimer's amyloid-beta peptide, leptin, insulin-like growth factor-I, and also regulates macromolecule transport from the cerebral microvessels and choroid plexus to cerebral ventricles, thereby maintaining homeostasis in cerebrospinal fluid (CSF), as well as exerting neuroprotective action [4547]. In the absence of Dab2, the protein levels of megalin are reduced and the subcellular localization of megalin is altered [48]. The choroid plexus starts to secrete CSF very early during development [49], correlating well with the early expression of Dab2 protein. We found that Dab2 is also prominently expressed in microvilli on the luminal surface of the embryonic choroid epithelium, consistent with a role associated with macromolecule transport and secretory functions. However, the change observed in asymmetric expression between the luminal and the basal surface from embryonic to adult states (i.e. more prominent expression in the basal compared to luminal aspect in the adult) remains unclear.

Dab2 immunoreactivity was detected in another developing circumventricular organ, the subcommissural organ (SCO). The SCO develops very early during embryogenesis, representing one of the first secretory brain structures to differentiate [50]. The very early ontogenetic differentiation of SCO secretory cells is crucial for the smooth circulation of CSF secreted from the choroid plexus during early development. The present study shows that Dab2 is specifically expressed in the developing SCO primordium at an early stage (E11.5). Both the choroid plexus and the SCO are secretory organs that develop very early in embryos. However, unlike the early and persistent expression of Dab2 in the choroid plexus, the Dab2 protein appeared transiently in SCO-secretory cells, a phenomenon that may possibly be related to the morphogenesis of SCO. Similar to the SCO, the developing pineal gland also showed transient Dab2 expression in the present study. Dab2 expression in the developing pineal gland was not detected at or before E14.5 but became distinct by E16.5, the stage at which pinealocytes undergo rapid proliferation in rodents [51]. It is likely that Dab2 is expressed in developing pinealocytes, although we cannot rule out the possibility that interstitial cells also express the Dab2 protein. The functional significance of a transient expression of Dab2 in the developing pineal gland remains to be investigated.

Other than the expression in secretory epithelial cells, Dab2 was also detected in cells of the myeloid lineage, specifically mononuclear phagocytes. We have clearly shown that Dab2 is expressed at early developmental stages in mononuclear phagocytes (Iba1+/Dab2+ cells) both outside the developing neural tube (from E9.5 onwards) and transiently within the neural tube (from E10.5 – E14.5). The expression of Dab2 (or p96) in mature macrophages is perhaps not surprising given its initial characterization from a macrophage cell line [1113]. However, the functional roles played by Dab2 in lineage commitment and differentiation of myeloid-derived mononuclear phagocytes are only recently being investigated [52], and have clear implications for determining the lineage and differentiation also of microglia. ICSBP (Interferon Consensus Sequence Binding Protein; a member of the interferon regulatory factor family of transcription factors which acts as a regulatory switch in myeloid differentiation [52]), and the Ets transcription factor PU.1, are haematopoietic-restricted transcription factors critical for the development of haematopoietic cells [52]. PU.1 is an essential factor required for myeloid cell differentiation [35]. Both of these transcription factors bind to the Dab2 promoter, but whereas ICSBP represses Dab2 promoter transactivation, PU.1 induces it [52]. Significantly, ICSBP represses the PU.1-induced transactivation, thereby suppressing Dab2 expression. These data indicate that cross-talk between PU.1 and ICSBP may be crucial in regulating genes related to myeloid cell differentiation and cell functions. Taken together and in context with our current findings showing an increased and persistent expression of Dab2 in developing and adult macrophages compared to the lack of Dab2 in adult microglia, these results allude to differences in myeloid cell differentiation (and/or commitment) between macrophages and microglial cells [35] that need to be explored in greater depth.

Transient expression of Dab2 was detected in early Iba-1+cells located within the neural tube, in cells closely apposed to blood vessels and to the ventricular walls. These observations may imply that at least a subpopulation of early microglia (Iba1+/Dab2+) are derived from myeloid progenitors migrating into neural tube via microvessels, rather than derived in situ [35]. Downregulation, or indeed lack of Dab2 expression in subsequently developing microglia may represent an important event during the differentiation and maturation of these cells.

Dab2 acts as a signalling molecule in CSF-1/MCSF (colony stimulating factor-1/macrophage colony stimulating factor) signal transduction [12, 13]. Microglia, unlike cells of the macrophage lineage, are not exclusively dependent on CSF-1 for cell survival and differentiation [5356]. The lack of Dab2 protein expression in microglia (Iba1+/Dab2-) which persists throughout development (E12.5-14.5 onwards in this study), in adult CNS microglia at rest (this study), and even under traumatic injury [57], may reflect this lack of dependency on CSF-1/MCSF for differentiation and/or survival of these cells, which in turn points to differences in environmental trophic support for mononuclear phagocytes within the CNS.

Although Dab2 was not expressed in adult microglia, it was expressed in another mononuclear phagocytic cell type in the CNS, namely perivascular macrophages which co-expressed F4/80 antigen. The differential expression of Dab2 in mononuclear phagocytes (microglia, fetal and adult macrophages and perivascular macrophages) renders it a useful marker to further study the origin and responses of these cells.

Conclusion

This is the first detailed report on the spatio-temporal expression of Dab2 during CNS development in mammals. Unlike Dab1, which is expressed in developing neuronal cells, Dab2 protein is dynamically expressed in various regions of the developing CNS, primarily restricted to non-neuronal cells including within circumventricular organs, in secretory cells, immature neuroepithelial cells, endothelial cells and in populations of mononuclear phagocytes (including fetal and perivascular macrophages), but lacking in neurons, neuroglial cells and in mature microglia. These findings are consistent with its expression pattern observed in the adult nervous system (this study and [57]), and in response to CNS injury [57] where Dab2 expression is primarily located to macrophages accumulating at the site of injury, but absent from neurons, neuroglia and microglia. Expression of Dab2 in polarized epithelial cells in developing circumventricular organs may be consistent with the well-documented functions of Dab2 in mediating endocytic trafficking of clathrin-coated vesicles [58, 59], and in directing cell surface positioning [60]. Dab2 is further implicated in myeloid cell differentiation and macrophage development. Our findings highlight the need for further investigations into the biological functions of Dab2 in early CNS and myeloid cell development.

Methods

Experimental animals

Random-bred ICR mice were kept at a constant temperature of 23 ± 2°C under a 12-hr light: 12-hr dark cycle at the Laboratory Animal Services Centre of the Chinese University of Hong Kong. They were provided with food and water ad libitum. All experimental procedures involving the use of animals for experimental purposes were performed under licence from the Government of the Hong Kong SAR and endorsed by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong (Animal Ethics Approval Ref. No. 04/006/ERG). Female mice weighing 35–40 g were paired with a male mouse overnight. The noon of the plug day was designated as embryonic day 0.5 (E0.5) assuming that copulation occurred around midnight.

Immunohistochemical staining and double immunofluorescence staining on tissue sections

Pregnant mice at different stages of pregnancy from E7.5 to E18.0 were sacrificed by cervical dislocation and uteri dissected open. For each developmental stage, at least 5–10 embryos were collected for analysis. Embryos were isolated, fixed in Bouin's fixative, and processed for wax sectioning. Sections of 7 μm thick were cut and mounted on Superfrost® Plus microscopic slides (VWR Scientific). Localization of the Dab2 protein by immunohistochemical staining was performed using the monoclonal anti-p96/Dab2 antibody (1:400, Transduction Laboratories/BD Biosciences). The peptide used to raise this monoclonal antibody corresponds to amino acids at positions 31–45 at the N-terminal of the full-length murine p96/Dab2; a domain that is shared by the three known isoforms of Dab2 (namely p96, p93 and p67) in the mouse. Antigen retrieval was carried out by microwave treatment in 0.1 M citric acid buffer (pH 6.0). Immunostaining was performed with the standard ABC method using DAB (Sigma) as the chromogen [2, 61]. Sections were counter-stained with hematoxylin, dehydrated in a graded series of alcohols, cleared in xylene and mounted in Permount (Fisher Scientific). Images were taken under a Zeiss microscope with a digital camera (Diagnostic instruments, USA). The specificity of p96/Dab2 was confirmed by (1) pre-adsorbing with the corresponding purified immunogen (BD Biosciences) for 6 hr at 4°C before adding to the sections, or (2) replacing the primary antibodies with PBS containing 5% normal horse serum. For double immunohistochemical localization, 4% paraformaldehyde fixed sections and antibodies to Dab2 (1:100, BD), Iba1 (1:75, Wako), F4/80 (1: 50, Serotec), PGP 9.5 (1: 800, Ultraclone) were used. Secondary antibodies used were Alexa Fluor-conjugated donkey antibodies against mouse, rabbit or rat (1:400, Invitrogen). Immunofluorescent images were captured using a confocal microscope (Olympus FluoView™-FV1000).

Whole mount immunohistochemistry

The procedure was carried out as previously described [62]. Briefly, embryos were collected and fixed in Bouin's fixative. They were dehydrated with upgraded methanol and stored in pure methanol at -20°C until use. Embryos were then treated with 2% Triton X-100 in DMSO/methanol (4:1) overnight for permeabilization. They were washed in 0.5% Triton X-100 in PBS (PBT) and treated with 1% periodic acid overnight to block endogenous peroxidase activity. Non-specific binding was blocked with 5% non-fat dry milk in PBT (PBTM) before incubating the embryos at 4°C overnight with the monoclonal anti-p96 antibody (1:400) [19, 24]. Signals were amplified with the standard ABC methods using DAB as chromogen. The entire neural tube was isolated using tungsten needles and brought to 80% glycerol in distilled water, through a graded series, to obtain transparency. Images were captured with an AxioCam (Zeiss) under a stereomicroscope (Zeiss).

Declarations

Acknowledgements

The work described in this paper was supported by grants from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project no. CUHK4262/04M and CUHK4412/06M), Dana-Farber/Harvard Cancer Center Ovarian Cancer SPORE grant P50CA165009 and R33CA103595 from National Institute of Health, Department of Health and Human Services.

Authors’ Affiliations

(1)
Department of Anatomy, Faculty of Medicine, The Chinese University of Hong Kong
(2)
Department of Rehabilitation Sciences, The Hong Kong Polytechnic University
(3)
Department of Gynecologic Oncology University of Texas M.D. Anderson Cancer Center T4.3908
(4)
Neuropathology Research Laboratory, Department of Life Sciences, Faculty of Science, The Open University
(5)
Department of Neuroscience, Institute of Psychiatry, King's College London

References

  1. Mok SC, Wong KK, Chan RK, Lau CC, Tsao SW, Knapp RC, Berkowitz RS: Molecular cloning of differentially expressed genes in human epithelial ovarian cancer. Gynecol Oncol. 1994, 52 (2): 247-252. 10.1006/gyno.1994.1040.View ArticlePubMedGoogle Scholar
  2. Mok SC, Chan WY, Wong KK, Cheung KK, Lau CC, Ng SW, Baldini A, Colitti CV, Rock CO, Berkowitz RS: DOC-2, a candidate tumor suppressor gene in human epithelial ovarian cancer. Oncogene. 1998, 16 (18): 2381-2387. 10.1038/sj.onc.1201769.View ArticlePubMedGoogle Scholar
  3. Fulop V, Colitti CV, Genest D, Berkowitz RS, Yiu GK, Ng SW, Szepesi J, Mok SC: DOC-2/hDab2, a candidate tumor suppressor gene involved in the development of gestational trophoblastic diseases. Oncogene. 1998, 17 (4): 419-424. 10.1038/sj.onc.1201955.View ArticlePubMedGoogle Scholar
  4. Tseng CP, Ely BD, Li Y, Pong RC, Hsieh JT: Regulation of rat DOC-2 gene during castration-induced rat ventral prostate degeneration and its growth inhibitory function in human prostatic carcinoma cells. Endocrinology. 1998, 139 (8): 3542-3553. 10.1210/en.139.8.3542.PubMedGoogle Scholar
  5. Schwahn DJ, Medina D: p96, a MAPK-related protein, is consistently downregulated during mouse mammary carcinogenesis. Oncogene. 1998, 17 (9): 1173-1178. 10.1038/sj.onc.1202038.View ArticlePubMedGoogle Scholar
  6. Fazili Z, Sun W, Mittelstaedt S, Cohen C, Xu XX: Disabled-2 inactivation is an early step in ovarian tumorigenicity. Oncogene. 1999, 18 (20): 3104-3113. 10.1038/sj.onc.1202649.View ArticlePubMedGoogle Scholar
  7. Zhou J, Scholes J, Hsieh JT: Characterization of a novel negative regulator (DOC-2/DAB2) of c-Src in normal prostatic epithelium and cancer. J Biol Chem. 2003, 278 (9): 6936-6941. 10.1074/jbc.M210628200.View ArticlePubMedGoogle Scholar
  8. Gertler FB, Bennett RL, Clark MJ, Hoffmann FM: Drosophila abl tyrosine kinase in embryonic CNS axons: a role in axonogenesis is revealed through dosage-sensitive interactions with disabled. Cell. 1989, 58 (1): 103-113. 10.1016/0092-8674(89)90407-8.View ArticlePubMedGoogle Scholar
  9. Gertler FB, Hill KK, Clark MJ, Hoffmann FM: Dosage-sensitive modifiers of Drosophila abl tyrosine kinase function: prospero, a regulator of axonal outgrowth, and disabled, a novel tyrosine kinase substrate. Genes Dev. 1993, 7 (3): 441-453. 10.1101/gad.7.3.441.View ArticlePubMedGoogle Scholar
  10. Hoffmann FM: Drosophila abl and genetic redundancy in signal transduction. Trends Genet. 1991, 7 (11-12): 351-355. 10.1016/0168-9525(91)90254-F.View ArticlePubMedGoogle Scholar
  11. Sheng Z, Smith ER, He J, Tuppen JA, Martin WD, Dong FB, Xu XX: Chromosomal location of murine disabled-2 gene and structural comparison with its human ortholog. Gene. 2001, 268 (1-2): 31-39. 10.1016/S0378-1119(01)00401-2.View ArticlePubMedGoogle Scholar
  12. Xu XX, Yang W, Jackowski S, Rock CO: Cloning of a novel phosphoprotein regulated by colony-stimulating factor 1 shares a domain with the Drosophila disabled gene product. J Biol Chem. 1995, 270 (23): 14184-14191. 10.1074/jbc.270.23.14184.View ArticlePubMedGoogle Scholar
  13. Jackowski S, Xu XX, Rock CO: Phosphatidylcholine signaling in response to CSF-1. Mol Reprod Dev. 1997, 46 (1): 24-30. 10.1002/(SICI)1098-2795(199701)46:1<24::AID-MRD5>3.0.CO;2-T.View ArticlePubMedGoogle Scholar
  14. Sheng Z, He J, Tuppen JA, Sun W, Fazili Z, Smith ER, Dong FB, Xu XX: Structure, sequence, and promoter analysis of human disabled-2 gene (DAB2). Genomics. 2000, 70 (3): 381-386. 10.1006/geno.2000.6383.View ArticlePubMedGoogle Scholar
  15. Hocevar BA, Smine A, Xu XX, Howe PH: The adaptor molecule Disabled-2 links the transforming growth factor beta receptors to the Smad pathway. Embo J. 2001, 20 (11): 2789-2801. 10.1093/emboj/20.11.2789.View ArticlePubMed CentralPubMedGoogle Scholar
  16. Zhou J, Hsieh JT: The inhibitory role of DOC-2/DAB2 in growth factor receptor-mediated signal cascade. DOC-2/DAB2-mediated inhibition of ERK phosphorylation via binding to Grb2. J Biol Chem. 2001, 276 (30): 27793-27798. 10.1074/jbc.M102803200.View ArticlePubMedGoogle Scholar
  17. Tseng CP, Huang CL, Huang CH, Cheng JC, Stern A, Tseng CH, Chiu DT: Disabled-2 small interfering RNA modulates cellular adhesive function and MAPK activity during megakaryocytic differentiation of K562 cells. FEBS Lett. 2003, 541 (1-3): 21-27. 10.1016/S0014-5793(03)00281-3.View ArticlePubMedGoogle Scholar
  18. Hocevar BA, Mou F, Rennolds JL, Morris SM, Cooper JA, Howe PH: Regulation of the Wnt signaling pathway by disabled-2 (Dab2). Embo J. 2003, 22 (12): 3084-3094. 10.1093/emboj/cdg286.View ArticlePubMed CentralPubMedGoogle Scholar
  19. Morris SM, Tallquist MD, Rock CO, Cooper JA: Dual roles for the Dab2 adaptor protein in embryonic development and kidney transport. Embo J. 2002, 21 (7): 1555-1564. 10.1093/emboj/21.7.1555.View ArticlePubMed CentralPubMedGoogle Scholar
  20. Cho SY, Cho SY, Lee SH, Park SS: Differential expression of mouse Disabled 2 gene in retinoic acid-treated F9 embryonal carcinoma cells and early mouse embryos. Mol Cells. 1999, 9 (2): 179-184.PubMedGoogle Scholar
  21. Hogan BL, Taylor A, Adamson E: Cell interactions modulate embryonal carcinoma cell differentiation into parietal or visceral endoderm. Nature. 1981, 291 (5812): 235-237. 10.1038/291235a0.View ArticlePubMedGoogle Scholar
  22. Smith ER, Capo-chichi CD, He J, Smedberg JL, Yang DH, Prowse AH, Godwin AK, Hamilton TC, Xu XX: Disabled-2 mediates c-Fos suppression and the cell growth regulatory activity of retinoic acid in embryonic carcinoma cells. J Biol Chem. 2001, 276 (50): 47303-47310. 10.1074/jbc.M106158200.View ArticlePubMedGoogle Scholar
  23. Morrisey EE, Musco S, Chen MY, Lu MM, Leiden JM, Parmacek MS: The gene encoding the mitogen-responsive phosphoprotein Dab2 is differentially regulated by GATA-6 and GATA-4 in the visceral endoderm. J Biol Chem. 2000, 275 (26): 19949-19954. 10.1074/jbc.M001331200.View ArticlePubMedGoogle Scholar
  24. Yang DH, Smith ER, Roland IH, Sheng Z, He J, Martin WD, Hamilton TC, Lambeth JD, Xu XX: Disabled-2 is essential for endodermal cell positioning and structure formation during mouse embryogenesis. Dev Biol. 2002, 251 (1): 27-44. 10.1006/dbio.2002.0810.View ArticlePubMedGoogle Scholar
  25. Prunier C, Howe PH: Disabled-2 (Dab2) is required for transforming growth factor beta-induced epithelial to mesenchymal transition (EMT). J Biol Chem. 2005, 280 (17): 17540-17548. 10.1074/jbc.M500974200.View ArticlePubMedGoogle Scholar
  26. Chan WY, Tam PP: A morphological and experimental study of the mesencephalic neural crest cells in the mouse embryo using wheat germ agglutinin-gold conjugate as the cell marker. Development. 1988, 102 (2): 427-442.PubMedGoogle Scholar
  27. Chan WY, Cheung CS, Yung KM, Copp AJ: Cardiac neural crest of the mouse embryo: axial level of origin, migratory pathway and cell autonomy of the splotch (Sp2H) mutant effect. Development. 2004, 131 (14): 3367-3379. 10.1242/dev.01197.View ArticlePubMedGoogle Scholar
  28. Tam PP, Beddington RS: The formation of mesodermal tissues in the mouse embryo during gastrulation and early organogenesis. Development. 1987, 99 (1): 109-126.PubMedGoogle Scholar
  29. Howell BW, Hawkes R, Soriano P, Cooper JA: Neuronal position in the developing brain is regulated by mouse disabled-1. Nature. 1997, 389 (6652): 733-737. 10.1038/39607.View ArticlePubMedGoogle Scholar
  30. Sanada K, Gupta A, Tsai LH: Disabled-1-regulated adhesion of migrating neurons to radial glial fiber contributes to neuronal positioning during early corticogenesis. Neuron. 2004, 42 (2): 197-211. 10.1016/S0896-6273(04)00222-3.View ArticlePubMedGoogle Scholar
  31. Olson EC, Kim S, Walsh CA: Impaired neuronal positioning and dendritogenesis in the neocortex after cell-autonomous Dab1 suppression. J Neurosci. 2006, 26 (6): 1767-1775. 10.1523/JNEUROSCI.3000-05.2006.View ArticlePubMedGoogle Scholar
  32. Huang CH, Cheng JC, Chen JC, Tseng CP: Evaluation of the role of Disabled-2 in nerve growth factor-mediated neurite outgrowth and cellular signalling. Cell Signal. 2007, 19 (6): 1339-1347. 10.1016/j.cellsig.2007.01.019.View ArticlePubMedGoogle Scholar
  33. Ito D, Imai Y, Ohsawa K, Nakajima K, Fukuuchi Y, Kohsaka S: Microglia-specific localisation of a novel calcium binding protein, Iba1. Brain Res Mol Brain Res. 1998, 57 (1): 1-9. 10.1016/S0169-328X(98)00040-0.View ArticlePubMedGoogle Scholar
  34. Ito D, Tanaka K, Suzuki S, Dembo T, Fukuuchi Y: Enhanced expression of Iba1, ionized calcium-binding adapter molecule 1, after transient focal cerebral ischemia in rat brain. Stroke. 2001, 32 (5): 1208-1215.View ArticlePubMedGoogle Scholar
  35. Chan WY, Kohsaka S, Rezaie P: The origin and cell lineage of microglia: new concepts. Brain Res Rev. 2007, 53 (2): 344-354. 10.1016/j.brainresrev.2006.11.002.View ArticlePubMedGoogle Scholar
  36. Rezaie P, Male D: Microglia in the fetal and adult human brain can be distinguished from other mononuclear phagocytes through their lack of CD163 expression. Neuroembryology. 2003, 3: 130-133. 10.1159/000074192.View ArticleGoogle Scholar
  37. Rezaie P: Microglia, from origins to differentiation, distribution and dynamics within the mammalian central nervous system. Trends in Glial Research - Basic and Applied. Edited by: Dheen ST, Ling EA. 2007, India , Research Signpost, 1-41.Google Scholar
  38. Barrow JR, Stadler HS, Capecchi MR: Roles of Hoxa1 and Hoxa2 in patterning the early hindbrain of the mouse. Development. 2000, 127 (5): 933-944.PubMedGoogle Scholar
  39. Lumsden A: The cellular basis of segmentation in the developing hindbrain. Trends Neurosci. 1990, 13 (8): 329-335. 10.1016/0166-2236(90)90144-Y.View ArticlePubMedGoogle Scholar
  40. Krumlauf R: Hox genes in vertebrate development. Cell. 1994, 78 (2): 191-201. 10.1016/0092-8674(94)90290-9.View ArticlePubMedGoogle Scholar
  41. Martinez-Ceballos E, Chambon P, Gudas LJ: Differences in gene expression between wild type and Hoxa1 knockout embryonic stem cells after retinoic acid treatment or leukemia inhibitory factor (LIF) removal. J Biol Chem. 2005, 280 (16): 16484-16498. 10.1074/jbc.M414397200.View ArticlePubMedGoogle Scholar
  42. Ericson J, Muhr J, Jessell TM, Edlund T: Sonic hedgehog: a common signal for ventral patterning along the rostrocaudal axis of the neural tube. Int J Dev Biol. 1995, 39 (5): 809-816.PubMedGoogle Scholar
  43. Marti E, Takada R, Bumcrot DA, Sasaki H, McMahon AP: Distribution of Sonic hedgehog peptides in the developing chick and mouse embryo. Development. 1995, 121 (8): 2537-2547.PubMedGoogle Scholar
  44. Oleinikov AV, Zhao J, Makker SP: Cytosolic adaptor protein Dab2 is an intracellular ligand of endocytic receptor gp600/megalin. Biochem J. 2000, 347 Pt 3: 613-621. 10.1042/0264-6021:3470613.View ArticlePubMedGoogle Scholar
  45. Dietrich MO, Spuch C, Antequera D, Rodal I, de Yebenes JG, Molina JA, Bermejo F, Carro E: Megalin mediates the transport of leptin across the blood-CSF barrier. Neurobiol Aging. 2007Google Scholar
  46. Chun JT, Wang L, Pasinetti GM, Finch CE, Zlokovic BV: Glycoprotein 330/megalin (LRP-2) has low prevalence as mRNA and protein in brain microvessels and choroid plexus. Exp Neurol. 1999, 157 (1): 194-201. 10.1006/exnr.1999.7052.View ArticlePubMedGoogle Scholar
  47. Carro E, Spuch C, Trejo JL, Antequera D, Torres-Aleman I: Choroid plexus megalin is involved in neuroprotection by serum insulin-like growth factor I. J Neurosci. 2005, 25 (47): 10884-10893. 10.1523/JNEUROSCI.2909-05.2005.View ArticlePubMedGoogle Scholar
  48. Nagai J, Christensen EI, Morris SM, Willnow TE, Cooper JA, Nielsen R: Mutually dependent localization of megalin and Dab2 in the renal proximal tubule. Am J Physiol Renal Physiol. 2005, 289 (3): F569-76. 10.1152/ajprenal.00292.2004.View ArticlePubMedGoogle Scholar
  49. Dziegielewska KM, Ek J, Habgood MD, Saunders NR: Development of the choroid plexus. Microsc Res Tech. 2001, 52 (1): 5-20. 10.1002/1097-0029(20010101)52:1<5::AID-JEMT3>3.0.CO;2-J.View ArticlePubMedGoogle Scholar
  50. Rodriguez EM, Rodriguez S, Hein S: The subcommissural organ. Microsc Res Tech. 1998, 41 (2): 98-123. 10.1002/(SICI)1097-0029(19980415)41:2<98::AID-JEMT2>3.0.CO;2-M.View ArticlePubMedGoogle Scholar
  51. Calvo JL, Boya J, Carbonell AL, Garcia-Maurino JE: Time of origin of the rat pineal gland cells. A bromodeoxyuridine immunohistochemical study. Histol Histopathol. 2004, 19 (1): 137-142.PubMedGoogle Scholar
  52. Rosenbauer F, Kallies A, Scheller M, Knobeloch KP, Rock CO, Schwieger M, Stocking C, Horak I: Disabled-2 is transcriptionally regulated by ICSBP and augments macrophage spreading and adhesion. Embo J. 2002, 21 (3): 211-220. 10.1093/emboj/21.3.211.View ArticlePubMed CentralPubMedGoogle Scholar
  53. Rezaie P, Male D: Differentiation, ramification and distribution of microglia within the central nervous system examined. Neuroembryology. 2002, 1: 29-43. 10.1159/000051020.View ArticleGoogle Scholar
  54. Witmer-Pack MD, Hughes DA, Schuler G, Lawson L, McWilliam A, Inaba K, Steinman RM, Gordon S: Identification of macrophages and dendritic cells in the osteopetrotic (op/op) mouse. J Cell Sci. 1993, 104 ( Pt 4): 1021-1029.Google Scholar
  55. Wiktor-Jedrzejczak W, Gordon S: Cytokine regulation of the macrophage (M phi) system studied using the colony stimulating factor-1-deficient op/op mouse. Physiol Rev. 1996, 76 (4): 927-947.PubMedGoogle Scholar
  56. Sasaki A, Yokoo H, Naito M, Kaizu C, Shultz LD, Nakazato Y: Effects of macrophage-colony-stimulating factor deficiency on the maturation of microglia and brain macrophages and on their expression of scavenger receptor. Neuropathology. 2000, 20 (2): 134-142. 10.1046/j.1440-1789.2000.00286.x.View ArticlePubMedGoogle Scholar
  57. Moon C, Lee J, Ahn M, Shin T: Involvement of Disabled-2 protein in the central nervous system inflammation following experimental cryoinjury of rat brains. Neurosci Lett. 2005, 378 (2): 88-91. 10.1016/j.neulet.2004.12.016.View ArticlePubMedGoogle Scholar
  58. Eden ER, Sun XM, Patel DD, Soutar AK: Adaptor protein disabled-2 modulates low density lipoprotein receptor synthesis in fibroblasts from patients with autosomal recessive hypercholesterolaemia. Hum Mol Genet. 2007, 16 (22): 2751-2759. 10.1093/hmg/ddm232.View ArticlePubMedGoogle Scholar
  59. Kowanetz K, Terzic J, Dikic I: Dab2 links CIN85 with clathrin-mediated receptor internalization. FEBS Lett. 2003, 554 (1-2): 81-87. 10.1016/S0014-5793(03)01111-6.View ArticlePubMedGoogle Scholar
  60. Yang DH, Cai KQ, Roland IH, Smith ER, Xu XX: Disabled-2 is an epithelial surface positioning gene. J Biol Chem. 2007, 282 (17): 13114-13122. 10.1074/jbc.M611356200.View ArticlePubMedGoogle Scholar
  61. Chan WY, Xia CL, Dong DC, Heizmann CW, Yew DT: Differential expression of S100 proteins in the developing human hippocampus and temporal cortex. Microsc Res Tech. 2003, 60 (6): 600-613. 10.1002/jemt.10302.View ArticlePubMedGoogle Scholar
  62. Lee YM, Osumi-Yamashita N, Ninomiya Y, Moon CK, Eriksson U, Eto K: Retinoic acid stage-dependently alters the migration pattern and identity of hindbrain neural crest cells. Development. 1995, 121 (3): 825-837.PubMedGoogle Scholar

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